The outlook and treatment of adverse human gamete interaction has drastically changed with the advent of intra-cytoplasmic sperm injection (ICSI) and manipulation of enriched but often poor sperm suspensions, sometimes yielding less than double digit counts. Palermo G D, Cohen J, Alikani M, Adler A, and Rosenwaks, Z., "Intracytoplasmic sperm injection: a novel treatment for all forms of male factor infertility", Fertil. Steril., 63, 1231-1240 (1995); Silber, S. J., Van Steirteghem, A. C., Liu, J., Nagy, Z., Tournaye, H. and Devroey, P., "High fertilization and pregnancy rate after intracytoplasmic sperm injection with spermatozoa from testicle biopsy", Hum. Reprod., 10, 148-152 (1995a).
Men who are azoospermic can now be treated using surgical isolation of sperm cells from their testicles or reproductive tract. Palermo, G., Joris, H., Devroey, P., and Van Steirteghem, A., "Pregnancies after intracytoplasmic sperm injection of single spermatozoon into an oocyte", Lancet, 340, 17-18 (1992); Craft, I., Bennett, V. and Nicholson, N. (1993), "Fertilising ability of testicular spermatozoa", Lancet, 342, 864 (1992); Schoysman, R., Vanderzwalmen, P., Segal-Bertin, G., and van de Casseye, M., "Successful fertilization by testicular spermatozoa in an in-vitro fertilization programme", Hum.Reprod., 8, 1339-1340 (1993); Silber, S. J., Nagy, Z., Liu, J., Tournaye, H., Lissens, W., Ferec, C., Liebaers, I., Devroey P. and Van Steirteghem, A. C., "The use of epididymal and testicular spermatzoa for intracytoplasmic sperm injection: the genetic implications for male infertility", Hum. Reprod., 10, 2031-2043 (1995b); Devroey, P., Liu, J., Nagy, Z., Goossens, A., Tournaye, H., Camus, H. and Van Steirteghem, A., "Pregnancies after testicular sperm extraction and intra-cytoplasmic sperm injection in non-obstructive azoospermia", Hum. Reprod., 10, 1457-1460 (1995). This can even include men with arrested spermiogenesis, since it is evident that spermatids contain all necessary elements for decondensation and complete participation at syngamy. Fishel, S., Aslam, I. And Tesarik, J., "Spermatid conception: a stage too early, or a time too soon?", Hum. Reprod. 11, 1371-1376 (1996).; Palermo, G., Munne, S. and Cohen, J., "The human zygote inherits its mitotic potential from the male gamete", Hum. Reprod., 9, 1220-1225 (1994).
In the near future even less mature diploid stages such as secondary spermatocytes and possibly spermatogonia may be used for fertilization or zygote reconstitution; this seems an obvious development, since in some men, spermatogenesis arrests early during meiosis. The in-vitro culture of spermatogonia appears to be hypothetically solved, but clinical application faces major physiological hurdles involving combinations of experimental molecular and cellular strategies that have not yet been developed.
Technical, physiological and genetic problems have already occurred in the new field of single mature gamete manipulation. The presence of Y-deletions in some azoospermic men and possible consequences in male offspring, has recently caused a review and change in regulations by The Dutch Health Council (1996). Health Council of the Netherlands (1996) Committee on in vitro fertilization, "Assisted fertilization: ICSI", The Hague. Publication no. 1996/06E. See also, Cummins, J. M. and Jequier, A. M., "Concerns and recommendations for intracytoplasmic sperm injection (ISCI) treatment", Hum. Reprod., 10 Suppl1: 138-143 (1995).
Another important problem concerns the possible negative consequences certain diagnostic and therapeutic extraction procedures may have on testicular function. Jarow, J., "Intratesticular arterial anatomy", J.Androl., 11, 255-259 (1990); Schlegel, P., "Physiologic consequences of TESE, Hum.Reprod., 11, abstract 159 (1996). Repeated surgical procedures are not only costly and invasive, but in the case of testicular sperm extraction (TESE), can cause transient and even permanent adverse physiologic effects. Schlegel, P., "Physiologic consequences of TESE", Hum.Reprod., 11, abstract 159 (1996). Repetition of these procedures can, in some cases, be avoided by cryopreservation of spermatozoa, but is only possible when sufficient numbers of functional cells are isolated. Silber, S. J., Van Steirteghem, A. C., Liu, J., Nagy, Z., Tournaye, H. and Devroey, P., "High fertilization and pregnancy rate after intracytoplasmic sperm injection with spermatozoa from testicle biopsy", Hum. Reprod., 10, 148-152 (1995a). Although there are anecdotal reports, Patrizio, P., Ord, T., Balmaceda, J. P. and Asch, R. H., "Successful fertilization, pregnancy, and birth using epididymal sperm frozen 24 hours after conventional oocyte insemination", Fertil. Steril., 64, 863-865 (1995); Podsiadly, B. T., Woolcott, R. J., Stanger, J. D. and Stevenson, K., "Case report: pregnancy resulting from intracytoplasmic sperm injection of cryopreserved spermatozoa recovered from testicular biopsy", Hum. Reprod., 11, 1306-1308 (1996), describing sperm survival and birth after cryopreservation of sperm-rich epididymal and testicular aspirations, conventional sperm freezing cannot work for limited numbers of sperm cells. Hewitt J, Cohen J, Mathew T and Rowland G F, "Cryopreservation of semen in patients with malignant disease: role of in vitro fertilization", Lancet, 2, 445-446 (1985). Yet it is crucial that a sperm freezing method be developed to avoid repeated surgical attempts at sperm extraction.
Therefore, in cases of very low sperm recovery from azospermic men, a method is needed for preservation and recovery of the sperm so that the necessary surgical procedures are not unnecessarily performed.
In addition, the problems of preserving and recovering single cells or very small biological samples occur in other fields, both scientific and medical. As a single cell is isolated, such as a hematopoetic stem cell, for use in autotransplantation into a patient, the number of cells isolated may be small. Since the procedure typically calls for a period of chemotherapy and/or radiation therapy, the stored cells must be preserved, for example frozen, for autotransplantation after therapy has completed.
Other fields of science and industry also require the isolation and preservation of single cell samples, or small groups of cells.
It is known to use empty zona pellucidae for the culturing of embryos from different species. In these known methods, a relatively large aperture, e.g., spanning about 280.degree., forms a flap in the wall of the zona through which a micropipette can be used insert embryonic material. In order to retain the embryonic material in the zona, and prevent attack by immune cells after insertion into the oviduct of a host animal, the zona is encased in an agar chip. Willadsen, S. M. "Nuclear Transplantation in Sheep", Nature, 320:63 (1986). Empty zona pellucidae have also been used for the storage of embryonic material for laboratory tests.
The zona pellucida is an extracellular coat that is synthesized by the oocyte and surrounds the egg and early embryo of all mammalian species. P. M. Wassarman: "Zona pellucida glycoproteins", Ann Rev Biochem 57, 414-442 (1988). The zona pellucida is normally the site of the initial interaction of the spermatozoa with the oocyte. This interaction includes the species-specific spermatozoa-zona pellucida binding and induction of the acrosome reaction (AR), both of which are prerequisites for successful in-vivo fertilization.
Results of cDNA cloning of the zona pellucida genes and analysis of the composition of zona pellucida from several different species indicate that zona pellucida is constituted of three or four sulfated glycoproteins. O. Epifano & J. Dean, "Biology and structure of the zona pellucida: a target for immunocontraception", Reprod Fertil Dev 6, 319-330 (1994). The zona pellucida of the mouse oocyte, one of the best studied zona pellucida, is composed of three sulphated glycoproteins termed ZP1, ZP2 and ZP3. P. M. Wassarman, "Zona pellucida glycoproteins", Ann Rev Biochem 57, 414-442 (1988); P. M. Wassarman, "Profile of a mammalian sperm receptor", Development 198, 1-17 (1990). ZP1 is a homodimer (Mr=185-200 kDa) and its chains are connected by intermolecular disulphide bonds. ZP2 (Mr=120-140 kDa) and ZP3 (Mr=83 kDa) form a heterodimer of long filaments with a repeating structure. P. M. Wassarman, "Zona pellucida glycoproteins", Ann Rev Biochem 57, 414-442 (1988); P. M. Wassarman, "Profile of a mammalian sperm receptor", Development 198, 1-17 (1990). ZP1 provides a structural integrity for the zona pellucida by cross-linking the ZP2/ZP3 filaments. Only ZP2 and ZP3 have been shown to possess biological functions. The mouse ZP1 is a 623 amino acid polypeptide chain with a signal peptide and a carboxyl terminal transmembrane domain, which is typical of all zona proteins. Epifano, O., et al., Development, 121.07:1947-1956.
ZP3 mediates the initial binding of acrosome-intact spermatozoa to the zona pellucida via O-linked side chains. H. M. Florman & P. M. Wassarman, "O-Linked oligosaccharides of mouse egg ZP3 account for its sperm receptor activity", Cell 41, 313-324 (1985); T. K. Rosiere & P. M. Wassarman, "Identification of a region mouse zona pellucida glycoprotein mZP3 that possesses sperm receptor activity", Dev Biol 154, 309-317 (1992). Following sperm binding, ZP3 induces the AR in the bound spermatozoa. J. D. Bleil & P. M. Wassarman, "Sperm-egg interactions in the mouse: sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein", Dev Biol 95, 317-324 (1983). The acrosome-reacted spermatozoa, which can no longer interact with ZP3, bind to ZP2 and penetrate through the zona pellucida. J. D. Bleil, J. M. Greve & P. M. Wassarman, "Identification of a secondary sperm receptor in the mouse egg zona pellucida: role for maintenance of binding of acrosome reacted sperm to eggs", Dev Biol 128, 376-385 (1988). After fertilization, there are molecular changes in ZP2 and ZP3 that constitute a block to polyspermy. ZP3 is converted to a form called ZP3.sub.f, which no longer binds acrosome-intact spermatozoa and is incapable to induce the AR. J. D. Bleil & P. M. Wassarman, "Sperm-egg interactions in the mouse: sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein", Dev Biol 95, 317-324 (1983). Since O-linked carbohydrates are implicated in interaction of ZP3 with spermatozoa, and there is no apparent change in the electrophoretic mobility of ZP3.sub.f, J. D. Bleil & P. M. Wassarman, "Sperm-egg interactions in the mouse: sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein", Dev Biol 95, 317-324 (1983); J. D. Bleil & P. M. Wassarman, "Mammalian sperm-egg interaction: Identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm", Cell 20, 873-882 (1980), this change in ZP3 is thought to be caused by a cortical granule-released glycosidase. D. J. Miller, X. Gong, G. Decker & B. D. Shur, "Egg cortical granule N-acetylglucosaminidase is required for the mouse zona block to polyspermry", J Cell Biol 123, 1431-1440 (1993). ZP2 is converted to a form called ZP2.sub.f that no longer interacts with acrosome-reacted spermatozoa. J. D. Bleil & P. M. Wassarman, "Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm", J Cell Biol 102, 1393-1371 (1986). ZP2 is cleaved by a protease from the cortical granules, C. C. Moller & P. M. Wassarman, "Characterization of a proteinase that cleaves zona pellucida glycoprotein ZP2 following activation of mouse eggs", Dev Biol 132, 103-112 (1989), and is detected by a shift in its electrophoretic mobility (from Mr=120 kDa to Mr=90 kDa) under reducing conditions. J. D. Bleil, C. E. Beoll & P. M. Wassarman, "Mammalian sperm-egg interaction: fertilization of mouse eggs triggers modification of the major zona pellucide glycoprotein, ZP2", Dev Biol 86, 189-197 (1981).
There are also reports regarding the composition of the human zona pellucida. S. Bercegeay, M. Jean, H. Lucas & P. Barriere, "Composition of human zona pellucida as revealed by SDS-PAGE after silver staining", Mol Reprod Dev 41, 355-359 (1995); J. Moos, D. Faundes, G. S. Kopf & R. M. Schultz, "Composition of the human zona pellucida and modifications following fertilization", Hum Reprod 10, 2467-2471 (1995); R. K. Naz & K. Ahmad, "Molecular identities of human sperm proteins that bind human zona pellucida: nature of sperm-zona interaction, tyrosine kinase activity, and involvement of FA-1", Mol Reprod Dev 39, 397-408 (1994); R. B. Shabanowitz & M. G. O'Rand, "Characterization of the human zona pellucida from fertilized and unfertilized eggs", J Reprod Fert 82, 151-161 (1988); R. B. Shabanowitz, "Mouse antibodies to human zona pellucida: evidence that human ZP3 is strongly immunogenic and contains two distinct isomer chains", Biol Reprod 43, 260-270 (1990). Shabanowitz and colleagues reported only two components (Mr=90-110 kDa and Mr=57-73 kDa) under nonreducing conditions and three components (Mr=90-110 kDa, Mr=65-78 kDa, and Mr=57-73 kDa) under reducing conditions. R. B. Shabanowitz & M. G. O'Rand, "Characterization of the human zona pellucida from fertilized and unfertilized eggs", J Reprod Fert 82, 151-161 (1988); R. B. Shabanowitz, "Mouse antibodies to human zona pellucida: evidence that human ZP3 is strongly immunogenic and contains two distinct isomer chains", Biol Reprod 43, 260-270 (1990). They termed these proteins ZP1, ZP2 and ZP3, respectively. A zona pellucida component corresponding to the mouse ZP1 (Mr=200 kDa) was not detected. Similarly, under reducing conditions Bercegeay et al. found protein components of 80-92 kDa, 58-66 kDa, and 54-72 kDa. S. Bercegeay, M. Jean, H. Lucas & P. Barriere, "Composition of human zona pellucida as revealed by SDS-PAGE after silver staining", Mol Reprod Dev 41, 355-359 (1995). These components likely correspond to mouse ZP2 and ZP3, respectively, based on their molecular weights. Naz and Ahmad showed that the human zona pellucida analyzed under non-reducing conditions exhibited 3 major protein bands of 220, 110 and 55 kDa. The zona pellucida protein that reacted strongest with the sperm proteins was the 55 kDa molecular region (ZP3). R. K. Naz & K. Ahmad, "Molecular identities of human sperm proteins that bind human zona pellucida: nature of sperm-zona interaction, tyrosine kinase activity, and involvement of FA-1", Mol Reprod Dev 39, 397-408 (1994). Recently, Moos et al., using a non-radioactive biotinylation- and a lectin-based detection system, found that, under non-reducing conditions, the human zona pellucida of unfertilized eggs is composed of three glycoprotein species designated as ZP1 (Mr.about.150 kDa), ZP2 (Mr.about.100 kDa) and ZP3 (Mr.about.55-65 kDa). J. Moos, D. Faundes, G. S. Kopf & R. M. Schultz, "Composition of the human zona pellucida and modifications following fertilization", Hum Reprod 10, 2467-2471 (1995).
In all the above studies, ZP1 was not detected after fertilization. In contrast, in the mouse, ZP1 is present and is apparently unaltered following fertilization. J. D. Bleil, C. E. Beoll & P. M. Wassarman, "Mammalian sperm-egg interaction: fertilization of mouse eggs triggers modification of the major zona pellucide glycoprotein, ZP2", Dev Biol 86, 189-197 (1981). Therefore, it has been suggested that in humans, the cortical granule-derived proteases may degrade ZP1 to forms that are not detectable by the electrophoresis process used in the studies. J. Moos, D. Faundes, G. S. Kopf & R. M. Schultz, "Composition of the human zona pellucida and modifications following fertilization", Hum Reprod 10, 2467-2471 (1995). See, Morales, P. and Llanos, M., "Interaction of human spermatozoa with the zona pellucida of oocyte: development of the acrosome reaction", Frontiers in Bioscience, 1, d146-160 (Aug. 1, 1995).
An artifical zona pellucida is disclosed in U.S. Pat. No. 5,272,086. This artificial zona is formed of a crosslinked microporous hydrogel, for implantation of a blastomere in the uterus of an animal species different from the blastomere species. In addition, purified zona pellucida proteins or glycoproteins are known. See, U.S. Pat. Nos. 4,801,689, and 4,847,363.